The objective is to develop methods for efficiently replacing or correcting mutant genes by homologous recombination with a wild type gene, or fragment thereof, newly introduced into a host cell. The immediate aim is to use fragments of a cloned human gene encoding adenine phosphoribosyltransferase (APRT) as a model system for rescuing resident mutant human APRT genes. The advantages of the APRT system are several: 1) we have cloned the human APRT gene and determined its nucleotide sequence; 2) the gene encodes a selectable marker which allows one to select either Aprt+ or Aprt- cells, depending upon the culture conditions used; 3) the functional gene is small, less than 3kb, and is therefore easy to manipulate; 4) there are several cell lines available that have mutant APRT genes; and 5) there are, in the human population, individuals that lack APRT activity due to mutations in their APRT genes. The strategy we initially propose is to construct vectors containing either the 5' or 3' half of the functional APRT gene. The vectors will also carry a gene encoding G418 resistance and a SupF marker. The vectors will be linearized within an intron and individually introduced into non-reverting Aprt- cells that carry a non-functional resident gene with a wild-type restriction pattern as determined by Southern blot analysis. We will select for Aprt+ cells and determine whether these cells arise as a consequence of homologous recombination. The criterion for recombination will be the appearance of a new but predicted restriction fragment. Once gene rescue by homologous recombination is established in this system, we will have the basis for testing methods that may increase the frequency of the event.